PROJECT SUMMARY Inflammatory bowel diseases (IBD), including Crohn?s disease and ulcerative colitis, affect an estimated 3.1 million U.S. adults. IBD pathophysiology is complex and involves genetic, environmental, and immune responses as well as alterations in gut microbial short-chain fatty acid (SCFA) levels. Recent studies from the Magness lab in murine ileal organoids demonstrated that exposure to IL-22 favors progenitor cell expansion over intestinal stem cell (ISC) self-renewal, and this phenotype is thought be regulated by activating Signal Transducer and Activator of Transcription (STAT)-3. Further, new evidence has shown that the gut microbial SCFA, butyrate, enhances IL-22 receptor expression and IL-22-induced activation of STAT3 in human colon cancer cells. While mouse, organoid, and cancer cell lines have helped to advance the field, more physiologically relevant in vitro human models are needed to increase accuracy and resolution of experiments aimed at predicting and uncovering the response of human gut ISCs and differentiated epithelium to such IBD-relevant factors. My aims will use a novel planar crypt-microarray (PCM) device and reporter cell lines to live-image primary human colonic stem cells during their response to IBD-related perturbations. PCMs have already been optimized for culture of mouse ISCs, and preliminary data demonstrates human ISC compatibility. Aim 1 will adjust factors such as PCM membrane composition, thickness, and timing of apical/basal media changes to promote confluent monolayers and compartmentalization of stem (Olfm4-EGFP) and differentiated (Muc2-BFP) cell zones. Using human-optimized PCMs, I will test the central hypothesis that butyrate-stimulated IL-22 signaling enhances asymmetric division and differentiation in human colonic ISCs. Aim 2 will use CRISPR/Cas9 indels to induce a frameshift loss-of-function (LOF) mutation in IL-22 receptor subunit alpha 1 into a tetracycline-inducible H2B::RFP (tetO-H2B::RFP) human colon stem cell line to test whether IL-22 signaling is necessary for asymmetric division. The tetO-H2B::RFP fusion protein allows for controlled, sensitive analysis of cell division dynamics in mammalian cells, with rapidly dividing cells corresponding to diluted RFP fluorescence intensity over time. RFP label-retaining cells will be assessed for phosphorylated STAT3 (pSTAT3) via immunofluorescence imaging on unsorted cell monolayers and for perturbed lineage pathways by FACS-scRNA-seq. The LOF model will be compared against IL22RA1+ controls on PCMs to determine if asymmetric division is enhanced by butyrate-stimulated IL-22 signaling. Our lab already has preliminary data for Olfm4-EGFP and H2B reporter lines. In the outstanding training environment in Dr. Scott Magness? group at the University of North Carolina at Chapel Hill, I will gain expertise in microfabrication and intestinal stem cell biology and learn new techniques in primary human colon stem cell culture, single-cell biology/genomics, and bioinformatics. My career development training plan focuses on research skills, professional development, and clinical skills to promote my path to independence as an academic physician-scientist.